Cell Cycle Activation During Imbibition and Visible Germination in Embryos and Megagametophytes of Jack Pine (Pinus banksiana Lamb.)

Flow cytometric determination of cell cycle activation duringimbibition and visible germination in five families of jackpine (Pinus banksiana Lamb.) embryos and megagametophytes revealedthat in seeds that had undergone no imbibition the majorityof cells were in the 2C state. As the imbibition period increased,less of the nuclei were blocked in the G0/G1 state and morebecome active in the cell cycle. The augmentation in the nucleiactive in the 2C–4C cycle as well as those with DNA levelshigher than the 4C state occured gradually and preceeded radicleemergence. In megagametophyte tissue examined at various stagesof imbibition, cell cycle activity became apparent rapidly followingimbibition. In nuclei of green and white embryos examined separatelythe ²frequency distributions were significantly different forall three families after 144h. As imbibition period increased,fewer nuclei from the green embryos were blocked in the 2C state,and more became active in the 2C–4C cell cycle. This wasnot the case for white embryos where no significant linear relationwas noted. Cell cycle activity in the hypocotyl+cotyledons regionand the emerging radicle were examined separately. Functionalrelations found in the hypocotyl+cotyledons region were notevident in the radicle. As visible germination proceeded, cellcycle activity in the hypocotyl + cotyledons region for thisperiod of germination showed a reversal of the activity notedduring imbibition: fewer nuclei were active and once again ahigher proportion were found in the 2C state. cell cycle; C levels; DNA content; flow cytometry; germination; imbibition; jack pine; megagametophyte; Pinus banksiana Lamb     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

<Emphasis Type="Italic">In Vitro</Emphasis> and cellular responses of jack pine embryos to three imbibition parameters

Summary Eighteen imbibition treatments with differing parameters of light conditions, temperature and duration were applied to jack pine seeds. After imbibition, embryos were excised and cultured in a liquid medium for 4 wk under continous agitation. At the end of the culture period, the embryos were classified according to five categories: nodule-forming, callus-forming, nodule plus callus-forming, white (non-responsive), and necrotic. Our results showed variation in the in vitro responsiveness of the embryos for the three parameters of imbibition and interactions among them. Three statistical models were tested to analyze the data on nodule formation, and two of them showed that the interaction between time and temperature, and between time and the combined variable temperature/light, were significant, while the light and duration variables had a significant effect only as single factors. The different morphogenic responses observed might indicate specific metabolic requirements of the embryos for the different developmental pathways. Afterwards, nine imbibition treatments were selected to evaluate the effects of the three parameters on the cell cycle and absolute DNA amount per nucleus. Following imbibition the number of cells in the G0–G1 phase decreased compared to the cells in dry seed, while the number of cells increased in the G2 phase after all the treatments except one. The percentage of cells in the different cell cycle phases varied significantly among the treatments. DNA amount fluctuated from 35.5 to 40.37 pg per nucleus. Compared to dry seeds with 40.19 pg DNA per nucleus, embryos from four imbibition treatments showed a pronounced decrease in the DNA by 5.9–11.8%. This might indicate underreplication of DNA sequences and reflect DNA plasticity with regards to imbibition and environmental factors.     

DNA CONTENT AND HETEROCHROMATIN VARIATIONS IN VARIOUS TISSUES OF PEANUT (ARACHIS HYPOGAEA)

The average 2C DNA amount for the peanut (Arachis hypogaea L.) genome is 4.21 pg, and 73% of the dormant peanut cotyledon nuclei displayed 8C DNA amounts or higher, as compared to 0 to 4% in root-shoot apices and leaf tissue. Thermal melt profiles and heterochromatin values indicated replication of the whole genome. Cotyledon nuclear DNA declined in the percent of polyploid nuclei as well as DNA amounts within ploidy classes during germination. The presence of high DNA C levels in cotyledons generated during embryogeny is interpreted to increase the protein-synthesizing capacity and subsequently supplies a ready source of nucleosides and phosphates during early embryo growth as a result of DNA degradation. However, the later DNA decline at the onset of cotyledon senescence was age related similarly to leaf senescence. The change in proportion of heterochromatin was related to the metabolic state of the tissue and not to the DNA content, as dormant and senescing nuclei contained a higher proportion of heterochromatin as compared to nuclei from metabolically active tissue such as germinating roots. The shift in heterochromatin is interpreted to be involved in gene expression.     

Idiosyncratic phenomenon of regeneration from cotyledons in the idiot fruit tree,Idiospermum australiense

Idiospermum australiense (Diels) S. T. Blake, is a rare species that produces very large diaspores (up to 225 g) comprising 2–6 cotyledons. Anecdotal evidence suggested that each cotyledon within the diaspore could develop an independent root/shoot axis in natural populations and under glasshouse conditions. To investigate this potential, 13 diaspores were collected. Six diaspores were kept intact and cotyledons in the remaining seven were separated, planted into soil and scored for germination. All shoots were allowed to grow for 12 weeks. To test for differences in growth response from seedlings emerging from intact diaspores and single cotyledons, plant height, shoot mass, stem mass, leaf mass and total leaf area were compared between groups using both absolute values and values standardized to initial reserve mass. To examine the effects of reserve mass in more detail, height to stem mass, leaf mass to stem mass and specific leaf area was also compared between groups. All intact cotyledons and 24 of the 27 single cotyledons produced a functional root/shoot system. Time to germination was not different between groups. Shoot mass, stem mass and leaf mass were significantly greater in intact diaspores than in single cotyledons, although no difference was found in any parameter when standardized to initial reserve mass. Shoots arising from individual cotyledons were significantly taller per gram initial weight, because of a significantly greater relationship between height and stem mass. Shoots arising from single cotyledons also had a significantly greater investment in leaf mass per unit stem mass, even though there was no difference in specific leaf area between groups. Thus, seedlings arising from individual cotyledons grew relatively taller and produced a greater area of leaf tissue relative to stem than those arising from intact diaspores.     

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

A comparison of internal standards for plant cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

DNA and protein content relationship in the cells of mature cotyledons ofPisum sativum

Summary The relationship between the DNA and reserve protein contents have been investigated in the cells of mature cotyledons ofPisum sativum. The study was made on two seeds chosen, on preliminary analysis, as having a markedly different protein content per cotyledon. The seed with the higher protein content was of theCameor variety, the other was of theAmino variety. The DNA content per cell was measured with a scanning microspectrophotometer and the protein content per cell using morphometric methods. In both seeds, nuclei of the storage parenchyma were polyploid in a range from 8 to 64 C levels, but the average DNA content per cell was higher in the Cameor specimen. The protein content also, whether expressed per unit of protoplasm or per cell, was always higher in the Cameor seed. The greater protein content of this seed was due to a higher content of protein per cell. In both seeds the level of ploidy and the reserve protein content per cell increased in the same way from the peripheral zone to the centre of the cotyledon. A high positive correlation was found for the two seeds between the DNA content and the protein content per cell.     

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

Age-related DNA changes in soybean ( Glycine max L. cv. Ransom) cotyledon nuclei were investigated by Feulgen cytophotometry and cloned DNA probes. The amount of nuclear DNA in 17-day-old senescing cotyledons was 23% lower than that of 5-day-old young cotyledons. In order to understand the nature of senescence-related DNA loss, fragments of repetitive DNA from young cotyledons were cloned into Escherichia coli HB101 cells by DNA recombination. The cloned DNA probed changes in specific repeated nucleotide sequences in senescing cotyledons. The colony hybridization between cloned DNA and [³²P]-labeled total DNA from 5- and 17-day cotyledons indicated loss of specific repeated sequences. The selected sequences of repeated DNA further showed a loss in their copy numbers. The study suggested that some repetitive DNA sequences were degraded selectively in the genome of senescing cotyledons.     

赛黑桦不同半同胞家系种子活力比较

种子活力能够预测幼苗的生长势和适应性,可作为评价采种母树的重要指标。赛黑桦作为东北林区木质坚硬的树种之一,关于其种子活力研究尚未见报道。比较不同家系赛黑桦种子活力,对赛黑桦采种母树进行初步评价,可为赛黑桦优良家系的选择提供参考。本研究以赛黑桦蛟河、集安和珲春3个采种地共30个半同胞家系为试材,对采种地及家系间种子千粒重、发芽率、发芽势、发芽指数、活力指数、出苗率和幼苗健壮率等7个性状进行了方差分析、遗传力估算、多重比较、相关性分析,并利用主成分分析法对参试家系进行了综合评价。结果表明,赛黑桦种子活力指数和幼苗健壮率在不同采种地间的差异达到了显著或极显著水平;7个性状在不同家系间的差异均达到显著或极显著水平;除出苗率和幼苗健壮率外,各性状的家系遗传力均高于0.68;7个性状间均呈现显著或极显著的正相关;采用主成分分析法选择JH6、JA1、JA7、JH10、JH3、HC1和HC10等7个家系为优良家系,这些家系的种子千粒重、发芽率、发芽势、发芽指数、活力指数、出苗率和幼苗健壮率的平均值分别比总平均值高3.87%、30.29%、30.22%、31.99%、58.22%、11.88%和30.60%。初步认为这些优良家系的母树是赛黑桦采种的首选母树。     

Influence of Pericarp,Cotyledon and Inhibitory Substances on Sharp Tooth Oak (Quercus aliena var. acuteserrata) Germination

In order to explore the mechanism of delayed and uneven germination in sharp tooth oak (Quercus aliena var. acuteserrata) (STO), mechanical scarification techniques were used to study STO root and shoot germination and growth. The techniques used were: removing cup scar (RS), removing the pericarp (RP), and cutting off 1/2 (HC) and 2/3 (TC) cotyledons. Germination percentage and root and shoot length for Chinese cabbage (Beassica pekinensis) seeds (CCS) were also investigated for CCS cultivated in a Sanyo growth cabinet watered by distilled water and 80% methanol extracts from the acorn embryo, cotyledon and pericarp with concentrations of 1.0 g, 0.8 g, 0.6 g and 0.4 g dry acorn weight per ml methanol. The results showed that the majority of roots and shoots from acorns with RP and HC treatment emerged two weeks earlier, more simultaneously, and their total emergences were more than 46% and 28% higher, respectively. TC accelerated root and shoot emergence time and root length, but root and shoot germination rate and shoot height had no significant difference from the control. Positive consequences were not observed on all indices of RS treatment. The germination rates of CCS watered by 1.0 g·ml⁻¹ methanol extracts from the embryo and cotyledon were significantly lower than those from the pericarp, and all concentrations resulted in decreased growth of root and shoot. Methanol extracts from pericarp significantly reduced root length of CCS, but presented little response in germination percentage and shoot length. The inhibitory effect was gradually increased with the increasing concentration of the methanol extract. We conclude that both the mechanical restriction of the pericarp and the presence of germination inhibitors in the embryo, cotyledon and pericarp are the causes for delayed and asynchronous germination of STO acorns.     

The Effects of Cell Cycle Parameters on Cell Wall Regeneration and Cell Division of Cotton Protoplasts (Gossypium hirsutum L.)

Protoplasts of cotton cotyledons were isolated and culturedto undergo cell wall regeneration and cell division. DNA contentand cell cycle parameters of nuclei from cotyledons and/or protoplastswere determined by flow cytometry. The DNA content of cotton,Gossypium hirsutum L., was estimated to be 4·34±0·12pg DNA per nucleus. There was a strong positive correlation between G₂ or Sand G₂,and cell wall regeneration and cell division and a strong negativecorrelation between G₁, and cell wall regeneration and celldivision of cotton cotyledon protoplasts. The cell cycle statusof cotyledons changes during their development; as the cotyledonsenlarge, the proportion of cells in G₀ and G₁ phases of thecell cycle increases. The implication of these results in relationto protoplast growth and development is discussed. Key words: Cell cycle parameters, cell wall regeneration, cell division, flow cytometry, Gossypium     

Transient gene expression of microprojectile-introduced DNA in Douglas-fir cotyledons

Summary Plasmid DNA containing the reporter gene uidA encoding -glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was introduced on high-velocity microprojectiles into cultured cotyledons of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. Transient gene expression was measured by counting the number of distinct loci of GUS activity per cotyledon. Contrary to published results on angiosperms, repeated bombardments did not increase expression in Douglas-fir. Expression varied significantly among cotyledons from different seedlings. The amount of time between DNA delivery and treatment of cotyledons with auxins and cytokinins strongly affected GUS expression. The optimal cytokinin pretreatment produced an average of 20 loci per cotyledon. In several experiments, more than 95% of the treated cotyledons exhibited at least some transient expression. Expression remained constant up to three days following DNA delivery into cotyledons.Abbreviations GUS -glucuronidase - CMV cauliflower mosaic virus - NOS nopaline synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzylaminopurine - X-GLUC 5-bromo-4-chloro-3-indolylglucuronide - kb kilobases Paper No. 2568 of the Forest Research Laboratory, Oregon State University, Corvallis, OR 97331, USA     

Some chemical properties of isolated pea nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

Some Chemical Properties of Isolated Pea Nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

A Comparison of Internal Standards for Plant Cytophotometry

Plant cell nuclei were compared with chicken erythrocyte nuclei for use as internal standards for microspectrophotometry. The amount of DNA per nucleus and the coefficient of variation for measurement of individual nuclei were determined for cells from dormant embryos of Pinus taeda and Pinus coulteri, from onion root tips and from chicken erythrocytes. The chicken erythrocytes had the least variability and thus were best suited for use as a standard. Onion root tips were least suitable, with a coefficient of variation 2 1/2 times that of erythrocytes. Although onion root tips have been used as an internal standard in other studies, their mitotic activity, in contrast with the nonreplication of DNA of mature erythrocytes, is reflected in a broad distribution of nuclei with values in the 2C-4C range. Coulter pine mature embryos were at the 3C level, whether dry or hydrated, while loblolly pine embryos were in the 2C state. This confirms previous reports. The coefficient of variability for the pine embryo cells was 1 1/2 times that of erythrocytes for nonhydrated seeds and twice the erythrocyte value for hydrated seeds. The larger 2C values for pine (26 pg for P. taeda and 17 pg for P. coulteri) are closer to values expected for many plant species than the 3 pg level of the chicken erythrocytes. Dormant P. taeda embryo cells (2C) are suggested as an alternative where the experimental material has large DNA values and/or chicken erythrocytes are difficult to procure. Large sample size is recommended for the plant materials if they are to be used as internal standards in Feulgen cytophotometry.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997